Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids

Biosensors are powerful tools for detecting, real-time imaging, and quantifying molecules, but rapidly constructing diverse genetically encoded biosensors remains challenging. Here, we report a method to rapidly convert enzymes into genetically encoded circularly permuted fluorescent protein-based indicators to detect organic acids (GECFINDER). ANL superfamily enzymes undergo hinge-mediated ligand-coupling domain movement during catalysis. We introduce a circularly permuted fluorescent protein into enzymes hinges, converting ligand-induced conformational changes into significant fluorescence signal changes. We obtain 11 GECFINDERs for detecting phenylalanine, glutamic acid and other acids. GECFINDER-Phe3 and GECFINDER-Glu can efficiently and accurately quantify target molecules in biological samples in vitro. This method simplifies amino acid quantification without requiring complex equipment, potentially serving as point-of-care testing tools for clinical applications in low-resource environments. We also develop a GECFINDER-enabled droplet-based microfluidic high-throughput screening method for obtaining high-yield industrial strains. Our method provides a foundation for using enzymes as untapped blueprint resources for biosensor design, creation, and application.

The ANL family is quite large and comprises two major subclasses of enzyme: NRPS adenylation domains and acyl synthetases.The paper describes trying to generally engineer sensors in this family, although the most successful sensors all derive from the peptidyl carrier protein domain-containing NRPS adenylation domains.If the journal is amenable, it might help the clarity of the paper to move the description of some of the less successful engineering efforts to a supplemental note and focus the main text on protein engineering to describe the efforts that eventually resulted in the sensors used in the final applications.
There are several aspects of the data that are missing or need clarification.Specifically, the authors do not report the quantum yield or the brightness of the sensors, which has direct implications for how easy the sensors are to use.Additionally, since the sensors appear to complete half of their catalytic reaction, care should be taken in terms of using KD to describe their dose-response profiles to their ligands since the catalytic activity means that this is not a simple binding affinity.Did the dose-response profiles change over time?How long were the titrations incubated prior to measurement?Lastly, reported spectra of the sensors and their description require further clarification.Table 1 reports the excitation maximum of Phe3 to be 460 nm, yet a visual inspection of the spectra doesn't support this.Furthermore, some sensors appear to be ratiometric in excitation (GECFINDER-His2, 4CA), but are described in a confusing fashion.
The discussion is lacking in perspective that provides the reader with context to evaluate the sensors and their potential impact.For example, all of the demonstrations of the use of these sensor use them as purified reagents at relatively high concentrations.Yet, the authors suggest that in the future these sensors might be used to measure metabolites intracellularly.Not only were the sensors not demonstrated to be compatible with in cellulo applications, but a sensor for metabolites that consumes ATP seems disadvantageous for capturing metabolic dynamics.It would also be helpful to contextualize the use of a fluorescent protein, rather than a system that might allow for amplification or provide more photons.

Some small changes:
The acronym POTS needs to be defined in the text cpEGFP changed to cpFP in the middle of the text and CpFP in the legend of Figure 2. Please regularize.
Reviewer #2 (Remarks to the Author): Wang et al. apply the strategy of fusing cicularly permuted GFP to proteins that undergo a ligandinduced conformational changes to the ANL superfamily of enzymes.The novelty and impact of the present manuscript is in the application of this strategy to enzymes and the demonstration of its broad applicability.Several bionsensors could be generated using rational design and screening methods and applications in sensing metabolites in blood or in screening for overproducing strains for biotechnological applications is demonstrated in extensive work.
The manuscript is generally sound and well written, however there are a number of grammatical problems or types, however mostly easy to understand and correct.Also the figures are of high quality.

Mayor points
When trying to generate a GLU-sensor by inserting cpGFP into PpsA-GluA, the authors state that they generated a sensor that detected ATP, but not glutamate (Extended Fig. 1 a-c).Extended Fig. 1c indicates that this sensor would also detect Mg2+ ions.However, more importantly, the authors add Mg2+ and ATP to all assays.ATP is cosubstrate for the enzymatic reaction and it probably binds together with ATP.The authors do not describe to what extend the other sensors depend on the ATP and Mg2+ concentrations.This is important information that should be presented.While this may not be so relevant for the in vitro applications described by the authors, it would be relevant for potential in vivo applications.
The authors provide no information as to how the dose-response curves were fitted to obtain the specified KD values.The authors need to specify the exact mathematical formula used for fitting and specify for each curve which parameters have been obtained.Also the software should be stated.Some dose-response curves appear as if they may be fitted by a simple sigmoidal curve without apparent cooperativity, such as Fig. 3a.In others the fluorescence changes take place over a wider concentration range, indicating that a Hill coefficient was probably used for fitting.Fig. 3a is a dose-response curve that likely does not support Kd fitting and this fluorescense change may be generated by other mechanisms than the ligand-induced conformational change.In curves extended Fig. 2 e and i as well as extended Fig. 3 a and g the fit curves quickly go into saturation (become horizontal) at higher substrate concentration and I really wonder which equilibrium binding model generates such a binding curve.

Minor points
Concerning the ANL enzymes used to design the biosensors.I would suggest to avoid the term biocatalyst and only use the term enzyme instead."Biocatalyst" is usually (admittedly not always) used when referring to enzymes in a biotechnological sense for synthesis etc.
On page 4, line 73 the authors cite two papers to state that cpFP-based biosensors usually have wider dynamic ranges as FRET-based biosensors.Please check if the cited papers really describe a comparison of FRET-based biosensors and cpFP and if you want to confirm this statement.For a non-expert, one should better specify "...usually have wider dynamic ranges (concerning the fluorescence change) as..." as one might apply "dynamic range" also to the analyte concentration.In Table 2, "ND" is specified for the operating range and KD for GECFINDER-Leu1 and Ligand Phe.Explain if ND stands for not determined or not detectable and why this is the case, although deltaF/F0 is reported.
Reviewer #3 (Remarks to the Author): The manuscript from Wang et al., shows the repurposing of a common biosynthetic enzyme fold for a new utility as a biosensor.Specifically, the authors focus on enzymes of the ANL superfamily, which are known to undergo conformational rotation during the catalytic cycle.Insertion of circularly permutated GFP superfolder into the loop region between the adenylation N domain and the C domain or between the adenylation domain and carrier domain yields an enzyme that undergoes fluoresence shift in response to a substrate of choice.The idea is clever and may have some utility.While the current response requires low to high micromolar concentrations of substrate, this is something that can be improved upon in future iterations.Overall I am enthusiastic about this work but there are several points that need to be clarified in a revised version.Specifically, 1.I am confused why the repurposed catalysts do not turn over.The dose responses only show linear increases.The authors should clarify or provide a rationale for why this is so. 2 is entirely too confusing as drawn.Not all ANL enzymes have a PCP domain so the top have of the panels do not make sense in the context of panel A.

Figure
3. There should be a more robust discussion of signal/noise of the response, especially if the authors are going to propose this as a system for use in biology.

Reviewers' comments & the authors' replies
We gratefully thank the editor and all reviewers for their time spend making their constructive remarks and useful suggestions, which has significantly raised the quality of the manuscript and has enable us to improve the manuscript.Each suggested revision and comment, brought forward by the reviewers was considered and addressed accordingly.
Below the comments of the reviewers are point by point response and the revisions are indicated.

Reviewer #1:
The article by Wang, J. et al entitled, "Converting biocatalysts into biosensors: repurposing conformational changes in ANL superfamily enzymes to detect various acids" describes the screening and generation of a series of fluorescent biosensors comprised of an enzyme and a circularly permuted GFP.The authors then use these tools to measure phenylalanine and glutamic acid in point-of-care diagnostics as well as in a highthroughput screening method for industrially-relevant yeast strains.
The authors extensively explore a family of enzyme and develop several fluorescent biosensors for acids, a couple of which show moderate fluorescence contrast.In general, the data support the conclusions of the authors.
Reply: We sincerely appreciate the reviewer's positive comments.
The article however suffers from a convoluted description of the protein engineering, some missing data, and a lack of perspective in the discussion.
The ANL family is quite large and comprises two major subclasses of enzyme: NRPS adenylation domains and acyl synthetases.The paper describes trying to generally engineer sensors in this family, although the most successful sensors all derive from the peptidyl carrier protein domaincontaining NRPS adenylation domains.If the journal is amenable, it might help the clarity of the paper to move the description of some of the less successful engineering efforts to a supplemental note and focus the main text on protein engineering to describe the efforts that eventually resulted in the sensors used in the final applications.
Reply: We gratefully appreciate your valuable suggestion.We agree that the protein engineering section could be clarified to enhance readability.In the revised version, we have moved the description of less successful engineering efforts, including the expansion of GECFINDER ligand binding domain and the preliminary efforts in rational site selection for cpEGFP insertion, to Supplemental Note1 and focus the main text on the protein engineering efforts that led to the sensors used in the final applications.
There are several aspects of the data that are missing or need clarification.Specifically, the authors do not report the quantum yield or the brightness of the sensors, which has direct implications for how easy the sensors are to use.
Reply: We apologize for not reporting the quantum yield or brightness of the sensors.We understand the importance of this information for assessing the ease of use of the sensors.In the revised version, we provide the quantum yield data for the sensors to address this issue.Specifically, on line 312 of the manuscript, we added the following statement: "During the determination of quantum yield, we observed that the absolute quantum yield of the GECFINDER variants ranged from 0. Additionally, since the sensors appear to complete half of their catalytic reaction, care should be taken in terms of using KD to describe their doseresponse profiles to their ligands since the catalytic activity means that this is not a simple binding affinity.Did the dose-response profiles change over time?How long were the titrations incubated prior to measurement?
Reply: Thank you for your valuable feedback regarding the use of K d to describe the dose-response profiles of the biosensors.We have carefully considered your suggestion and have made the necessary revisions in the revised manuscript.We have replaced the use of K d with EC50 (halfmaximal effective concentration) to describe the dose-response profiles in the revised manuscript.EC50 represents the concentration of ligand at which half of the maximum response is achieved.
Regarding the stability and time-dependence of the dose-response curve, the dose-response curve is plotted after the sensor response reaches a stable state.Typically, the dose-response curve of the sensor stabilizes within 5 minutes after the addition of the analyte and remains unchanged thereafter.
Once the stable state is reached, there are no further changes observed over time.The measurements were initiated immediately after adding the substrate, and readings were taken every minute for a continuous period of 15 minutes.
We have incorporated this additional information regarding the determination of dose-response curves into the Methods section, specifically in lines 757-761."The dose-response curve was determined immediately after the addition of the analyte.The total duration of the measurement was 15 minutes, with readings taken at one-minute intervals.
The dose-response curve was plotted using the fluorescence values obtained after achieving stability in the fluorescence signal." These modifications will provide a more comprehensive understanding of the experimental procedures and ensure transparency in our data analysis.
Lastly, reported spectra of the sensors and their description require further clarification.Table 1 reports the excitation maximum of Phe3 to be 460 nm, yet a visual inspection of the spectra doesn't support this.
Reply: Thank you for your valuable comments and suggestions.Regarding the excitation maximum of GECFINDER-Phe3, we agree that based on the fluorescence spectra, the optimal excitation wavelength appears to be around 480 nm.However, as indicated in Table 1, the maximum ΔF/F 0 for GECFINDER-Phe3 is observed at 460 nm.Therefore, we selected 460 nm as the excitation wavelength for GECFINDER-Phe3.To address this discrepancy, we have added relevant explanatory notes in the Table 1 of manuscript, clarifying the rationale behind our choice of excitation wavelength.Specifically, "The excitation wavelength of GECFINDER- Phe is selected at the maximum ΔF/F 0 , not the wavelength at the maximum fluorescence intensity."Furthermore, some sensors appear to be ratiometric in excitation (GECFINDER-His2, 4CA), but are described in a confusing fashion.
Reply: Thank you for your feedback regarding the description of ratiometric sensors.We apologize for any confusion caused by the initial presentation of the data.We have revised the description of the excitation spectra for GECFINDER-4CA, and the updated details can be found in the manuscript from lines 196 to 200.Specifically, " However, the GECFINDER-4CA was ratiometric in excitation.The emission spectrum shown in Fig. 3 b was measured when the excitation wavelength was 450 nm.As a result, it exhibited an opposite trend compared to the doseresponse curve depicted in Fig. 3 a."We have provided a clearer and more coherent explanation to ensure a better understanding of the ratiometric behavior of GECFINDER-4CA.
The discussion is lacking in perspective that provides the reader with context to evaluate the sensors and their potential impact.For example, all of the demonstrations of the use of these sensor use them as purified reagents at relatively high concentrations.Yet, the authors suggest that in the future these sensors might be used to measure metabolites intracellularly.Not only were the sensors not demonstrated to be compatible with in cellulo applications, but a sensor for metabolites that consumes ATP seems disadvantageous for capturing metabolic dynamics.
It would also be helpful to contextualize the use of a fluorescent protein, rather than a system that might allow for amplification or provide more photons.
Reply: Thank you for raising your concerns regarding the compatibility of our sensors for intracellular applications and the limitations of using fluorescent proteins.We acknowledge your reservations about the application of these sensors within cells, and we agree that our current study has not demonstrated their suitability for intracellular use.Regarding your point about the limitations of using fluorescent proteins, we recognize that they have certain drawbacks.However, this research represents our initial work in developing sensors based on the ANL family, and fluorescent proteins is a simple and relatively easy solution and it show great results for our applications.Additionally, other systems such as bioluminescent systems, may have disadvantages such as potential interference in signal amplification or the requirement for additional substrates.Furthermore, the conformational changes in ANL enzymes may not be effectively transmitted to amplification systems.We also acknowledge the need to explore alternative reporting systems that can overcome the limitations of fluorescent proteins in terms of signal amplification and photon emission.In subsequent studies, we have already begun investigating other systems that can address these challenges and potentially provide improved performance.
Some small changes: The acronym POTS needs to be defined in the text Reply: Thank you for pointing out the missing definition of the acronym "POTS" in the manuscript.However, upon reviewing the manuscript, we could not find any instances where "POTS" is used as an abbreviation.We are uncertain if you might be referring to the abbreviation "POCT" instead.
If "POCT" is the intended abbreviation, we provide a specific explanation at line 77-78 of the manuscript.Specifically, "However, such methods lack the detection throughput capacity and simplicity that are urgently needed in many applications, such as point-of-care testing Reply: Thank you for your suggestion.We apologize for the confusion caused by using both "cpEGFP" and "cpFP" interchangeably.We have made the necessary adjustments to regularize the abbreviation throughout the text.We have corrected the legend of Figure 2 to ensure consistency.
Reviewer #2: Wang et al. apply the strategy of fusing cicularly permuted GFP to proteins that undergo a ligand-induced conformational changes to the ANL superfamily of enzymes.The novelty and impact of the present manuscript is in the application of this strategy to enzymes and the demonstration of its broad applicability.Several bionsensors could be generated using rational design and screening methods and applications in sensing metabolites in blood or in screening for overproducing strains for biotechnological applications is demonstrated in extensive work.
The manuscript is generally sound and well written, however there are a number of grammatical problems or types, however mostly easy to understand and correct.Also the figures are of high quality.
Reply: We sincerely appreciate the reviewer's positive comments.

Mayor points
When trying to generate a GLU-sensor by inserting cpGFP into PpsA-GluA, the authors state that they generated a sensor that detected ATP, but not glutamate (Extended Fig. 1 a-c).Extended Fig. 1c indicates that this sensor would also detect Mg2+ ions.However, more importantly, the authors add Mg2+ and ATP to all assays.ATP is cosubstrate for the enzymatic reaction and it probably binds together with ATP.The authors do not describe to what extend the other sensors depend on the ATP and Mg2+ concentrations.This is important information that should be presented.While this may not be so relevant for the in vitro applications described by the authors, it would be relevant for potential in vivo applications.
Reply: We appreciate your valuable suggestion.We agree that clarifying the sensor's dependence on ATP and Mg 2+ concentrations is crucial.In the revised manuscript, we have provided a detailed description of the impact The authors provide no information as to how the dose-response curves were fitted to obtain the specified KD values.The authors need to specify the exact mathematical formula used for fitting and specify for each curve which parameters have been obtained.Also the software should be stated.Some dose-response curves appear as if they may be fitted by a simple sigmoidal curve without apparent cooperativity, such as Fig. 3a.In others the fluorescence changes take place over a wider concentration range, indicating that a Hill coefficient was probably used for fitting.Reply: Thank you for bringing these concerns to our attention.We apologize for not providing detailed information regarding the fitting of dose-response curves.In the revised version, we have included the software, mathematical formula, and parameters used for fitting each curve.
All dose-response curves were fitted using the five-parameter model (Hill 5), with the formula y = A min + (A max -A min ) / (1 + (x 0 /x) ^h) ^s, and the specific details can be found in Extended Data 4.
Regarding the cooperativity issues of Fig. 3a, we have carefully reviewed your observation regarding Fig. 3a and its apparent lack of cooperativity.Regarding the curves in Extended Fig. 2e, 2i, and Extended Fig. 3a, 3g, where the fit curves quickly reach saturation at higher substrate concentrations.We speculate that this might be due to larger errors in the data points at the end of the dose-response curves.On the other hand, slightly altered pH values in the reaction system due to the higher substrate concentrations could lead to small variations in GECFINDER's fluorescence performance.Therefore, these factors may have contributed to the fit curves quickly reaching saturation at higher substrate concentrations and then drop a little at end.

Minor points
Concerning the ANL enzymes used to design the biosensors.I would suggest to avoid the term biocatalyst and only use the term enzyme instead.
"Biocatalyst" is usually (admittedly not always) used when referring to enzymes in a biotechnological sense for synthesis etc.
Reply: Thank you for your suggestion.We have made the appropriate adjustments in the manuscript by avoiding the use of the term 'biocatalyst' and using the term 'enzyme' instead to describe the ANL enzymes employed in our biosensor design.Reply: Thank you for your suggestion.We acknowledge that enzymes are inherently catalytic.We have removed the redundant term "catalytic" in the mentioned sentence on page 5, line 99.Please refer to the figure below for the specific details: Fig. 1 The GECFINDER screening method overview.The GECFINDER screening method can be roughly divided into 4 parts.The first part is the design of GECFINDER.After obtaining the ANL superfamily protein sequences, if there is no crystal structure, homology modeling is needed to determine the positions of hinge A and hinge B according to its three-dimensional structure, and cpEGFP with random linker is inserted into hinge A or hinge B.
The second part is the construction of GECFINDER random linker library.Firstly, the DNA sequence of ANL superfamily member is integrated into the expression plasmid, and then random linker primers are designed at the insertion position of cpEGFP, and the linear DNA fragment of LBD is obtained by PCR.The cpEGFP DNA fragment is integrated into the linear DNA fragments of LBD.The plasmid of GECFINDER random linker library is transferred into Escherichia coli to induce expression of GECFINDER random linker library.The third part is the screening of GECFINDER random linker library.The differences in fluorescence values of GECFINDER random linker library before and after the addition of ligand are detected by the microplate reader, and the effective GECFINDER is cultured, purified, and characterized.The fourth part is the various potential applications of GECFINDER including FADS screening, tissue cell imaging, POCT assay, etc.
Page 12, line 223: "By comparing the sequences of the GrsA-PheA with a known structure (PDB ID: 223 1AMU) and the By comparing the sequences other 160 A domains, the...": Replace "and the other" by "with".
Reply: We gratefully appreciate for your valuable comment.We have revised the sentence on page 13, line 240 to replace "and the other" with "with" for clarity and accuracy.
In Table 2, "ND" is specified for the operating range and KD for GECFINDER-Leu1 and Ligand Phe.Explain if ND stands for not determined or not detectable and why this is the case, although deltaF/F0 is reported.
Reply: We apologize for the confusion caused by the "ND" specification in the operating range and KD value for GECFINDER-Leu1.
GECFINDER-Leu1 showed an increase in fluorescence intensity upon the addition of Phe; however, the resulting ΔF/F 0 values were relatively low, leading to a low signal-to-noise ratio for the sensor.Consequently, fitting dose-response curves under such conditions would result in significant errors, making the determination of accurate operational range and KD values unreliable.Hence, we did not provide the operational range and KD value for GECFINDER-Leu1.We have now added relevant clarification to Table 2 in the manuscript.Specifically, "Due to the ΔF/F 0 values of GECFINDER-Leu1 for Phe being less than 0.26, the signal-to-noise ratio was low, resulting in significant errors when fitting dose-response curves.
As a result, the operating range and EC50 values for this sensor was not provided in this study." Reviewer #3: The However, when cpEGFP is inserted into Hinge A, the second half-reaction, which involves the C-terminal small subunit's active site replacing AMP with CoA, is significantly inhibited due to the altered spatial positioning between the N-terminal large subunit and C-terminal small subunit caused by cpEGFP insertion.As a result, turnover is not observed.Similarly, when cpEGFP is inserted into Hinge B, the relative spatial positioning between the PCP domain and A domain is affected, preventing the transfer of the substrate-AMP complex to the PCP domain, thus resulting in the absence of turnover.It is the insertion of cpEGFP that hinders the turnover process in GECFINDER, leading to a linear segment in the dose-response curve.
We hope this explanation clarifies the absence of turnover in the ANL enzymes and provides insight into the linear increases observed in the dose-response curves.Thank you for raising these questions, and if you have any further inquiries or concerns, please do not hesitate to let us know.3.There should be a more robust discussion of signal/noise of the response, especially if the authors are going to propose this as a system for use in biology.
Reply: We gratefully appreciate for your valuable comment.In the revised manuscript, we have expanded the discussion on signal-to-noise ratio, emphasizing the importance of this parameter for the practical application of the biosensors.Specifically, in lines 643-646 of the manuscript, we have added the following content: "Additionally, achieving a high signal-tonoise ratio poses another challenge for intracellular application.Although some GECFINDER variants (Ile, His, Pro) currently exhibit lower signalto-noise ratios, we have shown that protein engineering can rapidly improve the performance of various GECFINDERs."We have also included considerations for optimizing the signal-to-noise ratio in future studies.
4. As I understand it, all of these engineered ANL enzymes require a concentration of ATP to maintain saturation.This is only briefly mentioned in the manuscript and should be stated more explicit.Moreover, where In light of this, the ongoing debate in our paper regarding dose-response curve fitting models centers around Extended Data Fig. 2 e, i and Extended Data Fig. 3 a-phe, g.These four curves exhibit a sudden flattening at the end.We have now fit these four curves and the normally shaped Fig. 3a using both the Hill4 and Hill5 models.Subsequently, an F-test was conducted to determine the more appropriate model for each case.The degrees of freedom, sum of squared residuals, and computed F-values for these five dose-response curves within both Hill4 and Hill5 models are listed in Table 1.
Table 1 At a significance level of 0.01, the computed critical F value is 0.162, and at a significance level of 0.05, it's 0.286.This implies that the computed F values in Table 1 are all greater than 0.162 and 0.286.Consequently, at both the 0.01 and 0.05 significance levels, it can be inferred that the Hill5 model outperforms the Hill4 model.This outcome suggests that the introduction of the additional parameter "s" significantly enhances the model's fit, leading to a better performance of the Hill5 model in interpreting the data.This underscores the necessity of introducing parameter "s" to quantitate asymmetry in the dose-response curve fitting of biosensors.
We hope this comprehensive explanation clarifies the rationale behind our choice of the Hill5 model and its statistical validation.If you have any further inquiries or concerns, please do not hesitate to reach out.My remarks in the first review concerning cooperativity were related to the slope of the dose-response curve in terms of apparent ligand binding cooperativity.
Reply: Thank you for your feedback.In the dose-response curve of GEFINDER-4CA, the slope is 0.86.If the slope is greater than 1, it indicates apparent ligand binding cooperativity.However, with a slope less than 1 in the dose-response curve of GEFINDER-4CA, it suggests the absence of apparent ligand binding cooperativity.

Fig. 1
Fig.1is a nice overview figure.I wonder if the authors could replace the figure of the instrument in "GECFINDER screening" by a scheme that illustrates the principle of the screening process.

(
POCT) scenarios."cpEGFP changed to cpFP in the middle of the text and CpFP in the legend of Figure 2. Please regularize.
of ATP and Mg 2+ on GECFINDER sensors.Please refer to lines 299-312 of the manuscript and Extended Data Fig.4for specific information regarding the ATP and Mg 2+ dependency of the sensors.Specifically, "We also investigated the dependence of GECFINDERs on Mg 2+ and ATP (Extended Data Fig.4).All GECFINDERs exhibited no significant change in fluorescence intensity in the presence of Mg 2+ alone.Among them, the fluorescence intensity of GECFINDER-4CA is slightly reduced upon the presence of ATP and Mg 2+ under 400 nm excitation, while the addition of 4-coumaric acid enhances the fluorescence intensity (Extended Data Fig. 4 a).This phenomenon corroborates with the distinct conformational changes observed in Hinge A of Nt4CL2 (Mg 2+ +ATP) (PDB ID: 5BSM) and Nt4CL2 (Coumaroyl-AMP) (PDB ID: 5BST) reported in the literature (Extended Data Fig. 4 k), where the direction of motion in Hinge A differs (ref.25).GECFINDER-Phe3/Pro/Ile3/Leu4 exhibited a slight increase in fluorescence intensity when both Mg 2+ and ATP were present (Extended Data Fig. 4 b, d, e, g).On the other hand, GECFINDER-Glu/His2/sβF/Tyr/Phe3.2 showed no significant change in fluorescence intensity when Mg 2+ and ATP were added (Extended Data Fig. 4 c, f, h, i, j)."Although the use of GECFINDER relies on the presence of cofactors Mg 2+ and ATP, their availability in the cellular environment is generally sufficient to support the functioning of GECFINDER.Therefore, the dependence on ATP and Mg 2+ is unlikely to have a significant impact on the intracellular application of GECFINDER.Extended Data Fig. 4 Dependence of GECFINDERs on Mg 2+ and ATP.a-j, Fluorescence intensities of GECFINDERs in the absence of any substrate, in the presence of Mg 2+ alone, ATP alone, both Mg 2+ and ATP, Mg 2+ and ATP with respective substrates.Mg 2+ concentration was 2.5 mM, and ATP concentration was 1 mM.a, 100 μM 4-coumaric acid, b, 100 μM phenylalanine, c, 1 mM glutamine, d, 1 mM proline, e, 100 μM isoleucine, f, 1 mM histidine, g, 1 mM leucine, h, 1 mM S-β-phenylalanine, i, 1 mM tyrosine, j, 1 mM phenylalanine.k, Conformational change of Nt4CL2 at Hinge A. All data shown are means ± S.D. (n=3).
Fig. 3a is a dose-response curve that likely does not support Kd fitting and this fluorescense change may be generated by other mechanisms than the ligand-induced conformational change.In curves extended Fig. 2 e and i as well as extended Fig. 3 a and g the fit curves quickly go into saturation (become horizontal) at higher substrate concentration and I really wonder which equilibrium binding model generates such a binding curve.

On page 4 ,
line 73 the authors cite two papers to state that cpFP-based biosensors usually have wider dynamic ranges as FRET-based biosensors.Please check if the cited papers really describe a comparison of FRETbased biosensors and cpFP and if you want to confirm this statement.For a non-expert, one should better specify "...usually have wider dynamic ranges (concerning the fluorescence change) as..." as one might apply "dynamic range" also to the analyte concentration.Reply: Thank you for your suggestion.We have carefully reviewed the two cited papers to confirm that they indeed describe a comparison between FRET-based biosensors and cpFP-based biosensors.In reference 13, it is stated, 'Compared with the FRET-based sensors which are ratiometric, single-FP-based intensiometric metal sensors have broader dynamic range and narrower excitation/emission wavelengths.'In reference 14, it is mentioned, 'Next, we intend to highlight the main advantages and disadvantages of cpFP-based probes over their counterparts, mainly FRET-based probes.Their most important benefit is the high dynamic range attributed to the efficient conformational coupling between sensory and reporter units, as described above.Moreover, even proteins with relatively modest conformational rearrangements can be tested as sensory units, providing an affordable signal-to-noise ratio for measurements.Second, they occupy a narrower part of the light spectrum, facilitating multiparameter imaging when several biochemical events are simultaneously monitored in a single living system.'Webelieve that both of these papers indicate that cpFP-based biosensors have a wider dynamic range and occupy a narrower part of the light spectrum compared to FRET-based biosensors.However, we agree with your suggestion to clarify that the wider dynamic range refers to the fluorescence change rather than the analyte concentration.In the revised manuscript, we have specifically emphasized that the wider dynamic range pertains to the fluorescence change.Thank you for bringing this to our attention, and we appreciate your careful review of the cited literature.Page 4, line 80: "Catalytic enzymes..." Enzymes are always catalytic

Fig. 1
Fig.1 is a nice overview figure.I wonder if the authors could replace the manuscript from Wang et al., shows the repurposing of a common biosynthetic enzyme fold for a new utility as a biosensor.Specifically, the authors focus on enzymes of the ANL superfamily, which are known to undergo conformational rotation during the catalytic cycle.Insertion of circularly permutated GFP superfolder into the loop region between the adenylation N domain and the C domain or between the adenylation domain and carrier domain yields an enzyme that undergoes fluoresence shift in response to a substrate of choice.The idea is clever and may have some utility.While the current response requires low to high micromolar concentrations of substrate, this is something that can be improved upon in future iterations.Overall I am enthusiastic about this work but there are several points that need to be clarified in a revised version.Specifically, Reply: We sincerely appreciate the reviewer's positive comments.1.I am confused why the repurposed catalysts do not turn over.The dose responses only show linear increases.The authors should clarify or provide a rationale for why this is so.Reply: We appreciate your inquiry regarding the absence of turnover in the ANL enzyme members utilized in our study.The ANL enzymes perform substrate binding and activation through a two-step catalytic reaction.In the first half-reaction, the substrate enters the binding pocket and consumes ATP to form a substrate-AMP intermediate.Insertion of cpEGFP into Hinge A or Hinge B does not significantly affect the catalytic performance of the N-terminal large subunit, probably allowing the ANL enzymes' first half-reaction to proceed largely unaffected in the GECFINDER system.

Table 2 .
" The detailed content of Extended Data Table2is as follows:

Table 1
Additionally, the GrsA A domain exhibits a K m value of 0.15 mM for ATP SS represents the sum of squared residuals, df indicates the degrees of freedom, and the subscripts 1 and 2 correspond to models with fewer and more parameters, respectively.Generally, if the computed F value surpasses a critical threshold, it signifies that the inclusion of the extra parameter (in this case, parameter "s") substantially enhances the model's fit.The choice of the critical value is often linked to the selected significance level, commonly 0.05 or 0.01.Therefore, if the calculated F value significantly exceeds the critical value, it can be concluded that the Hill5 model, relative to the Hill4 model, performs better in interpreting the data, and this improvement is statistically significant.However, if the F value is not significant, even with the introduction of more parameters in the Hill5 model, it cannot definitively prove its superiority in terms of fitting over the Hill4 model.The critical F values can be obtained by referencing an F-distribution table or by using statistical software.In software such as Excel, the F.INV() function can compute the critical F value.Computed for a significance level of 0.01, the critical F value for degrees of freedom 8 and 7 is 0.162, and for a significance level of 0.05, it's 0.286.
information on the Km value of ATP for Nt4CL2, a closely related homolog, At4CL2, has a reported ATP Km value of 0.163 mM (ref.35).where